The cellular response to ischemia and other forms of stress is a critical determinant of viability in the setting of vascular disease, and therefore affects the outcome of patients with stroke, myocardial infarction, and peripheral vascular disease. Cellular viability is affected in a negative fashion by stress-responsive signaling pathways that activate cell death programs and other deleterious processes, and in a positive fashion by protective responses, the most powerful and well- characterized of which are the molecular chaperones or heat shock proteins. We have recently identified a novel, developmentally regulated protein called CHIP (carboxyl terminus of Hsc 70- interacting protein) that interacts with the molecular chaperones (Hsc70, Hsp70, and Hsp90) and negatively regulates their functions. We have also shown that CHIP targets a subset of cellular proteins, including NOS3, for degradation and can activate the NF-kappaB signaling pathway. We hypothesize that CHIP antagonizes the cellular effects of molecular chaperones in tissues in which it is highly expressed, such as the heart. The study of CHIP should therefore provide an excellent model to understand protective mechanisms in the setting of ischemic vascular disease. To test our hypotheses, we will characterize the role of heat shock proteins and CHIP in regulation of stress-responsive signaling pathways such as NF- kappaB and NOS3 (Aims I and II) and determine the effects of modulation of CHIP expression on the cellular stress response (apoptosis, inflammation, and hypertrophy) in cell culture (Aim III). To define the role of CHIP in myocardial ischemia, we have created mice deficient in CHIP by homologous recombination. These mice will be utilized to determine the role of CHIP in modulating the response to hypertrophy and ischemia (Aim IV). These studies should help us to understand the role of molecular chaperones and in ubiquitin-proteasome pathway in cellular protective responses in the setting of ischemia, and have the potential to identify new targets for intervention in the treatment of ischemic cardiovascular and cerebrovascular disease.